Energy and nutrient utilisation by embryonic reptiles

Most reptiles are oviparous, with the developing embryos relying on the contents of the yolk to sustain development until hatching (lecithotrophy). The yolk is composed primarily of lipid and protein, which act as an energy source and the essential components to build embryonic tissue. Nevertheless, yolk and the resulting embryos contain many other nutrients, including inorganic ions, vitamins, carotenoids, water and hormones. Apart from water and oxygen, which may be taken up by eggs, and some inorganic ions that can come from the eggshell or even from outside the egg, everything required by the embryo must be in the egg when it is laid. Approximately 20% of squamate reptiles are viviparous, exhibiting a variety of placental complexities. Species with complex placentae have reduced yolk volumes, with the mother augmenting embryonic nutrition by provision across the placenta (placentotrophy). Despite assumed advantages of placentotrophy, only 5 out of approximately 100 lineages of viviparous squamates exhibit substantial placentotrophy. This paper reviews available and recent information on the yolk contents of a variety of squamate reptiles to ask the question, how are nutrients transported from the yolk to the embryo or across the placenta? Although, current available data suggest that, in broad terms, yolk is taken up by embryos without discrimination of the nutrients, there are some apparent exceptions, including the very long chain polyunsaturated fatty acids. In addition, fundamental differences in the patterns of energy utilisation in lizards and snakes suggest fundamental differences in lipid profiles in these taxa, which appear to reflect the differences between placentotrophic and lecithotrophic viviparous lizards.     

Maternal adjustment or constraint: differential effects of food availability on maternal deposition of macro‐nutrients,steroids and thyroid hormones in rock pigeon eggs

In oviparous species like birds, eggs provide the direct environment in which embryos are developing. Mothers may adjust different egg components in different ways in reaction to environmental cues either to adjust offspring development or because of constraints. In this study, we investigated the effects of food quality and quantity before and during egg laying on three different aspects of egg quality: macro‐nutrients (egg and yolk mass), androgens (testosterone and androstenedione), and thyroid hormones (3,5,3′‐triiodothyronine, T3 and l ‐thyroxine, T4), using the rock pigeon (Columba livia). As expected, egg and yolk mass were significantly reduced for the eggs laid under the poor‐food condition, indicating a maternal trade‐off between offspring and self in allocating important resources. We did not find any significant change in yolk testosterone or their within‐clutch pattern over the laying sequence. This is consistent with the fact that, in contrast with nutrients, these hormones are not costly to produce, but does not support the hypothesis that they play a role in adjusting brood size to food conditions. In contrast, we found that T3 levels were higher in the egg yolks under the poor‐food condition whereas the total T4 content was lower. This change could be related to the fact that iodine, the critical constituent of thyroid hormones, might be a limiting factor in the production of this hormone. Given the knowledge that food restriction usually lead to reduction of circulating T3 levels, our results suggested that avian mothers can independently regulate its concentrations in their eggs from their own circulation. The study demonstrates that environmentally induced maternal effects via the egg can be a result of a combination of constrained resources and unconstrained signals and that thyroid hormones might be an interesting case of both. Therefore, this hormone and the interplay of different maternal effects on the offspring phenotype deserve much more attention.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Yolk testosterone varies with sex in eggs of the lizard,Anolis carolinensis

In the green anole (Anolis carolinensis), a lizard with genotypic sex determination, yolk testosterone (T) concentration is greater in male-producing than female-producing eggs at oviposition, but the source and potential effects were not clear from previous studies. If yolk T levels are also sex-specific before eggs are laid, a period during which embryonic steroidogenesis is unlikely, it would strongly suggest that the difference in yolk T is maternally derived. We collected yolk samples from eggs shelling within the oviducts of anesthetized females, and then allowed these females to lay the eggs naturally. Eggs were incubated to hatching to determine sex morphologically, and yolk T concentrations were analyzed by radioimmunoassay. As is the case just after they are laid, yolk T is higher in male than female oviductal eggs. To our knowledge, this is the earliest sex difference reported for any yolk steroid. We suggest that maternally derived yolk T levels could influence sex by differentially affecting male- and female-inducing sperm, because fertilization occurs after yolk deposition and ovulation, while the egg is in the oviduct. Our results, together with those of an increasing number of studies, suggest that a relationship between hormones and vertebrate sex determination may be more widespread than generally appreciated.     

Quail Egg Yolk: A Novel Cryoprotectant for the Freeze Preservation of Poitou Jackass Sperm

For many years, attempts have been made to establish a sperm bank for the Poitou jackass population which is threatened with extinction. Unfortunately, no cryopreservation technique has ever been described for spermatozoa of this species. In an attempt to find a suitable technique, we studied the relative effectiveness of chicken egg yolk and quail egg yolk in preserving the motility and characteristics of movement of Poitou jackass spermatozoa during the freezing–thawing process. Semen was diluted to 60 × 10⁶sperm/ml in a preservation medium containing 4% (v/v) glycerol with 0, 2, 5, 10, 15, or 20% (v/v) of chicken or quail egg yolk. The chemical composition of these two eggs was compared. Effects were assessed using an automated analyzer which measured curvilinear velocity (VCL), straight line velocity (VSL), and the velocity of the average path. Linearity was defined as VSL/VCL × 100. The amplitude of the lateral head displacement was also measured. It was found that after the freeze–thaw process, quail egg yolk improved the percentages of motile and progressively undulating spermatozoa and the movement characteristics compared with chicken egg yolk. The optimal concentration of quail egg yolk was 10%. The general composition of the two types of egg yolk were similar, but quail egg yolk contained significantly more phosphatidylcholine, less phosphatidylethanolamine, and a smaller ratio of polyunsaturated to saturated fatty acids than chicken egg yolk. The improvement of motility for frozen–thawed Poitou jackass spermatozoa using frozen–thawed quail egg yolk compared to chicken egg yolk may be due to the differences in composition of the two yolks.     

Japanese quail selected for high plasma corticosterone response deposit high levels of corticosterone in their eggs

Poor habitat quality or body condition often correlates with high responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis rather than with elevated baseline levels of glucocorticoids. We hypothesized that, for egg-laying vertebrates, high responsiveness of the HPA axis would correspond to high concentrations of corticosterone in yolk. We tested the prediction that Japanese quail (Coturnix coturnix japonica) selected for high plasma corticosterone response to brief immobilization (HS quail) would lay eggs with higher yolk corticosterone concentrations than birds selected for low response (LS quail). Quail from both lines were left undisturbed, outside of the stressors associated with daily management, before a first round of egg collection. In a second experiment, quail of both lines were experimentally stressed during the week before egg collection. In both cases we found quail from the HS line to lay eggs with significantly higher yolk corticosterone concentrations than quail of the LS line. After exposure to added experimental stressors, the line difference was more pronounced (increasing from 62% to 96%). There was no line difference in concentrations of yolk testosterone. Our results suggest that (1) genetic differences underly differences in the transfer of maternal corticosterone to yolk and (2) females may be able to control deposition of corticosterone into yolk through a mechanism independent of baseline corticosterone titers.     

Negative effects of yolk testosterone and ticks on growth in canaries

Maternal yolk hormones in bird eggs are thought to adjust the offspring to the post-hatching environment. This implies that the effects of maternal yolk hormones should vary with the post-hatching environment, but to date such context-dependency has largely been ignored. We experimentally increased yolk testosterone concentrations in canary eggs and simultaneously manipulated the post-hatching context via an experimental tick-infestation of the chicks. This allows us to evaluate the context-dependency of hormone-mediated maternal effects, as it has previously been shown that ectoparasites alter the maternal yolk androgen deposition. The experimental tick infestation reduced growth in chicks from sham-treated eggs, indicating harmful effects of this ectoparasite in canaries. Chicks from testosterone-treated eggs were not affected in their development by ticks, suggesting lower ectoparasite vulnerability. But this may also be due to the fact that experimentally elevated yolk testosterone levels impaired growth even under parasite-free conditions. This contrasts previous studies, but these studies often manipulated first laid eggs, while we used eggs of subsequent laying positions. Later laid eggs are presumably of lower quality and contain higher yolk testosterone concentrations. Thus, the effects of elevated yolk testosterone on growth may be dose-dependent or vary with the egg quality, suggesting prenatal context-dependency.     

Genetic differences in yolk testosterone levels influence maternal hormone deposition in the second laying cycle in Japanese quails

Maternally-derived yolk androgens exhibit distinct among- and within-female variations but limited data refer to inter-seasonal changes of maternal hormones in the yolk. We investigated the deposition of yolk testosterone (T) across two laying cycles in Japanese quail. To test how genetically-determined differences influence between cycle variations in yolk androgens we compared females from low (LET) and high (HET) egg T lines at the end of the first and at the beginning of the second laying cycle after an induced moult. Line differences in yolk T levels exhibited high consistency exceeding two reproductive cycles. Yolk T concentrations increased in the second laying cycle in HET but not in LET females. Plasma T levels did not differ between cycles in both lines and no line differences were found either before or after the moult indicating the presence of mechanisms limiting the increase of T concentrations in the circulation. Differences in the yolk T levels were not accompanied by changes in the egg and yolk mass. The HET quail laid eggs with heavier eggshell than the LET quail. Our results demonstrate different abilities of mothers to deposit T in their eggs over two reproductive seasons with expected consequences on the development of their progeny.     

Maternally Derived Egg Hormones,Antibodies and Antimicrobial Proteins: Common and Different Pathways of Maternal Effects in Japanese Quail

Avian eggs contain a variety of maternally-derived substances that can influence the development and performance of offspring. The levels of these egg compounds vary in relation to environmental and genetic factors, but little is known about whether there are correlative links between maternal substances in the egg underlying common and different pathways of maternal effects. In the present study, we investigated genetically determined variability and mutually adjusted deposition of sex hormones (testosterone-T, androstenedione-A₄ and progesterone-P₄), antibodies (IgY) and antimicrobial proteins (lysozyme) in eggs of Japanese quail (Coturnix japonica). We used different genetic lines that were independently selected for yolk T concentrations, duration of tonic immobility and social reinstatement behaviour, since both selections for behavioural traits (fearfulness and social motivation, respectively) produced considerable correlative responses in yolk androgen levels. A higher selection potential was found for increased rather than decreased yolk T concentrations, suggesting that there is a physiological minimum in egg T levels. Line differences in yolk IgY concentrations were manifested within each selection experiment, but no consistent inter-line pattern between yolk IgY and T was revealed. On the other hand, a consistent inverse inter-line pattern was recorded between yolk IgY and P₄ in both selections for behavioural traits. In addition, selections for contrasting fearfulness and social motivation were associated with changes in albumen lysozyme concentrations and an inverse inter-line pattern between the deposition of yolk IgY and albumen lysozyme was found in lines selected for the level of social motivation. Thus, our results demonstrate genetically-driven changes in deposition of yolk T, P₄, antibodies and albumen lysozyme in the egg. This genetic variability can partially explain mutually adjusted maternal deposition of sex hormones and immune-competent molecules but the inconsistent pattern of inter-line differences across all selections indicates that there are other underlying mechanisms, which require further studies.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Absorption, transportation and digestion of egg white in quail embryos

The present study was done to reveal how egg white is taken up by embryonic tissues, the pathway through which egg white is transported, and the location where it is digested during the development of the quail Coturnix japonica. Antiserum against quail ovalbumin was raised in rabbit and used as a probe. By immunoelectron microscopy, the uptake of ovalbumin on a small scale by receptor-mediated endocytosis was observed in the ectodermal cells of the yolk sac on days four to seven of incubation. The uptake of egg white on a large scale by fluid-phase endocytosis took place in the cells generally referred to collectively as the 'albumen sac'. The ovalbumin was transported through the albumen sac into the extraembryonic cavity during days eight to 10, and then into the amniotic cavity through the amnion approximately on day 10. Ovalbumin was present in the intestinal lumen on days 11 and 14, but it was not digested in the intestinal epithelial cells. The ovalbumin was detected in the yolk of embryos after day 10. Immunoblot testing, as well as a fluoroimmunoassay, revealed that the location where the amount of ovalbumin was highest changed chronologically from the extraembryonic cavity on day 10 to the amniotic cavity on day 11, the intestinal lumen on day 12 and then to the yolk on day 13. Several low molecular proteins which cross-reacted with the antiserum were observed in the extracts of the yolk. The reaction producing these proteins depended on low pH (approximately 3.0) and was inhibited by pepstatin A. The ovotransferrin was similarly digested. These results indicate that egg white is, for the most part, transported through the albumen sac to the yolk via the extraembryonic cavity, the amniotic cavity, and the intestinal lumen, and is digested in the yolk by aspartic proteinases.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

No sex difference in yolk steroid concentrations of avian eggs at laying

Yolk steroids of maternal origin have been proposed to influence genetic sex determination in birds, based on sex differences in yolk steroid concentrations of peafowl eggs incubated for 10 days. More recent reports dispute this proposal, as yolk steroids in eggs incubated for 3 days do not show such sex differences. To date, research examining this phenomenon has only analysed incubated eggs, although sex in avian species is determined before incubation begins. This may be a serious methodological flaw because incubation probably affects yolk steroid concentrations. Therefore, we investigated sex differences in yolk steroid concentrations of unincubated avian eggs. We withdrew yolk for steroid analysis from fresh, unincubated Japanese quail (Coturnix japonica) eggs by biopsy, and then incubated those eggs for 10 days, after which we harvested the embryonic material for genetic sexing and the incubated yolk for further steroid analysis. We found no sex differences in fresh Japanese quail eggs; however, sex differences were apparent in yolk steroids by day 10 of incubation, when female eggs had significantly more oestrogen in relation to androgen than male eggs. Concentrations of all yolk androgens decreased dramatically between laying and day 10 of incubation, whereas oestradiol (E2) concentrations increased marginally. Thus, yolk concentrations of androgens and E2 do not appear critical for avian sex determination.     

Penguin Chicks Benefit from Elevated Yolk Androgen Levels under Sibling Competition

Crested penguins (genus Eudyptes) have a peculiar hatching pattern, with the first-laid egg (A-egg) hatching after the second-laid egg (B-egg) and chicks from A-eggs typically having a much lower survival probability. Maternal yolk androgens have been suggested to contribute to the competitive superiority of the B-chick in southern rockhopper penguins Eudyptes chrysocome, given their important role in mediating sibling competition in other species. We therefore increased the yolk androgen levels in freshly-laid eggs and examined the consequences for sibling competition - via effects on embryonic developmental times, chick growth and early survival. We placed one androgen-treated egg and one control egg into each foster nest, matching them for mass, laying date and laying order. The androgen treatment did not significantly affect embryonic developmental times or chick measurements at hatching. However, elevated yolk androgen levels benefitted chick growth in interaction with the number of siblings in a brood. Chicks from androgen-treated eggs had faster growth in the presence of a sibling than chicks from control eggs. Under these circumstances they also had a higher survival probability. Thus maternal androgens appear to reinforce the observed hatching pattern, facilitating brood reduction. This contrasts to most previous studies in other species where yolk androgens have been shown to compensate for the negative consequences of delayed hatching within the brood hierarchy.     

Maternal allocation strategies and differential effects of yolk carotenoids on the phenotype and viability of yellow-legged gull (Larus michahellis) chicks in relation to sex and laying order

Egg quality may mediate maternal allocation strategies according to progeny sex. In vertebrates, carotenoids have important physiological roles during embryonic and post-natal life, but the consequences of variation in yolk carotenoids for offspring phenotype in oviparous species are largely unknown. In yellow-legged gulls, yolk carotenoids did not vary with embryo sex in combination with egg laying date, order and mass. Yolk lutein supplementation enhanced the growth of sons from first eggs but depressed that of sons from last eggs, enhanced survival of daughters late in the season, and promoted immunity of male chicks and chicks from small eggs. Lack of variation in egg carotenoids in relation to sex and egg features, and the contrasting effects of lutein on sons and daughters, do not support the hypothesis of optimal sex-related egg carotenoid allocation. Carotenoids transferred to the eggs may rather result from a trade-off between opposing effects on sons or daughters.     

Availability of avidin-bound biotin to the chicken embryo.

Avidin, an exceptionally stable protein in egg white, binds the vitamin biotin with very high affinity and can induce biotin deficiency when fed to animals. To determine if biotin bound to avidin is available to the chicken embryo, the fate of [3H]biotin complexed to avidin was monitored during embryonic development. The majority (greater than 85%) of the [3H]biotin was extraembryonic until the day before hatching, when embryos swallow egg white and withdraw the yolk sac into their abdomen. Thus, biotin in the egg white of chicken eggs contributes little to the biotin status of the chick prior to hatching. After hatching, much of the [3H]biotin was assimilated. About 30% of the total was found in the liver and kidneys by 4 days of age. The biotin in liver was associated with large proteins and not with avidin. In a separate experiment, biotin injected into the egg white of biotin-deficient eggs failed to increase embryonic development or hatchability. Both experiments suggest that biotin in egg yolk is the primary and virtually sole source of biotin for the chicken embryo.     

Genetic parameters of egg characteristics in Japanese quail

Heritability, phenotypic and genetic correlations of egg characteristics of Japanese quail were investigated to obtain basal information on breeding, strain identification and genetic monitoring. For this study, 3230 eggs produced from 323 female were used. Measurement were taken on egg weight, yolk weight, albumen weight, shell weight, egg shape index, albumen height, specific gravity, shell thickness, shell strength and yolk color. Heritability estimates of egg characteristics were high and ranged from 0.62 to 0.84. Diverse phenotypic and genetic correlations were observed among egg characteristics. These results indicate that individual selection should be the most efficient method of improving egg characteristics and that consideration should be given to the interrelationship of characteristics to accomplish genetic improvement. The possibility exists these assays of egg characteristics can be used for strain identification and genetic monitoring without killing an individual of quail.     

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 °C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.     

Yolk androgens reduce offspring survival

Females may favour some offspring over others by differential deposition of yolk hormones. In American kestrels (Falco sparverius), we found that yolks of eggs laid late in the sequence of a clutch had more testosterone (T) and androstenedione (A4) than yolks of first-laid eggs. To investigate the effects of these yolk androgens on nestling 'fitness', we injected both T and A4 into the yolks of first-laid eggs and compared their hatching time, nestling growth and nestling survival with those of first-laid eggs in which we injected vehicle as a control. Compared to controls, injection of T and A4 at a dose intended to increase their levels to those of later-laid eggs delayed hatching and reduced nestling growth and survival rates. Yolk androgen treatment of egg 1 had no effect on survival of siblings hatching from subsequently laid eggs. The adverse actions of yolk androgen treatment in the kestrel are in contrast to the favourable actions of yolk T treatment found previously in canaries (Serinus canaria). Additional studies are necessary in order to determine whether the deposition of yolk androgens is an adaptive form of parental favouritism or an adverse by-product of endocrine processes during egg formation. Despite its adaptive significance, such 'transgenerational' effects of steroid hormones may have helped to evolutionarily shape the hormonal mechanisms regulating reproduction.     

Egg quality of Japanese quail depends on layer age and storage time

The aim of the study was to analyse egg quality changes of Japanese quail during the reproductive season and after several days of storage. A significantly higher egg weight was recorded in week 9 than in week 25 and week 31 of age ofbirds. The egg specific gravity value of eggs also decreased with the age of quails. The smallest yolk weight and its percentage proportion in egg mass were observed in week 25. Eggs laid in weeks 9 and 25 were characterised by similar and lower (P < or = 0.05) white weight in comparison to those examined on the last date. The highest white proportion in egg weight was recorded during the 25th week. The value of this trait was similar or smaller on the remaining dates. Similar and lower values of the white index and Haugh units, in comparison with the first date, were determined in eggs laid on weeks 25 and 31, whereas similar and greater eggshell thickness than in the 9th week of age was recorded on the last two dates of examination. Lower (P < or = 0.05) egg weight was observed after 5 and 8 than after 0 (measured on the day of laying) days of storage. Likewise, smaller egg specific gravity was recorded after three consecutive periods of storage in comparison with the eggs examined on the day of laying. Eggs examined after 3, 5 and 8 days of storage, in comparison with those stored for 0 days, were characterised by lower yolk index. Significantly smaller weight and white index than after 0 days were determined in eggs stored, respectively, for 5 and 8 as well as for 3, 5 and 8 days. Similarly, smaller numbers of Haugh units were determined after 3 consecutive storage periods in comparison with the eggs examined on the day of laying. Eggs stored for 0 and 3 days were characterised by a greater eggshell weight than those examined after 5 and 8 days. The weight of Japanese quail eggs decreased significantly from day 5 onwards, whereas the egg specific gravity-- from the 3rd day of storage. Yolk and white quality began to deteriorate already after 3 days of egg storage. It appears, therefore, that egg storage of Japanese quails before hatching lasting more than 3 days in the analysed conditions deteriorated the eggshell quality and internal egg traits.